Genetic modification or transformation is the technique wherein one or a few gene(s) are added to a commercial interesting genotype or clone. In principle a successful transformation system requires an efficient system where new plants are formed from specific plant parts (stem, leaf, node and so on) or from protoplasts (single cells without cell wall) derived of these parts, a system to transfer DNA molecules to the plant""s parts or protoplasts and a system to select tissue and plants which contain and express the introduced gene(s).
In principle protoplasts are the most ideal system for DNA delivery. They can be cultured as single cells that produce multicellular colonies from which plants develop. Plants derived from protoplasts are generally clonal in origin. This provides a useful tool for any transformation system, because it will eliminate chimerism in transgenic plants.
Cassava is very recalcitrant for plant regeneration of protoplasts. There is only one report of shoot regeneration from protoplasts of cassava (Shahin and Shephard, 1980). They used well expanded leaves for the isolation of protoplasts. Despite considerable efforts, plant regeneration from protoplasts (isolated from leaves, stems, and roots) has never been repeated since then (Anonymus, 1985; Nzoghe, 1991; Anthony et al., 1995, Sofiari, 1996). A logical approach was to use tissues which contain embryogenic cells. Such cells are found in the apical meristems, young leaves or somatic embryos cultured on auxin supplemented media (Stamp and Henshaw, 1987a; Raemakers et al., 1993a). However, protoplasts isolated from these tissues gave in the best case green callus and adventitious roots (Sofiari, 1996). Recently, a new type of somatic embryogenesis was developed. In this in vitro system the embryos do not develop beyond the (pre-)globular stage and the embryogenic callus is highly friable (Taylor et al., 1995). Transfer of this friable embryogenic callus (FEC) to liquid medium resulted in a suspension-like culture. In leek (Buitenveld and Creemers, 1994), petunia (Power et al., 1979), rice (Kyozuka et al., 1988), sugarcane (Chen, et al., 1988), and wheat (Chang et al., 1991) such cultures were an excellent source for protoplast regeneration.
We have now found that in cassava FEC is the only tissue from which protoplasts can be isolated which are able to regenerate into plants sofar.
Thus the present invention provides a method for producing protoplasts of cassava or a closely related species, which protoplasts are capable of regeneration into plants, comprising producing friable embryogenic callus from explants of cassava or a closely related species and isolating protoplasts from said friable embryogenic callus. It appears, as will be described below, that for obtaining suitable protoplasts the culture in solution of the FEC is quite important. Therefore the present invention further provides a method wherein the friable embryogenic callus is subjected to culture in a liquid medium.
Protoplasts are preferably produced by subjecting plant cells to enzymatic breakdown of the cell walls. The invention thus provides a method whereby a mixture of cell wall degrading enzymes, such as a cellulase, a pectolyase and/or a macerozyme are used to produce protoplasts.
It also appears that the method according to the invention works best when the plants from which explants are to be taken are pretreated. Therefore the invention provides a method whereby the plants from which explants are taken are pretreated with an auxin as described below.
On the explants preferably embryogenesis is induced resulting in an invented method whereby the friable embryogenic callus is produced from torpedo shaped primary or mature embryos. The reason is explained in the detailed description. Protoplasts obtainable by a method as disclosed above are also part of the invention.
An important reason for wanting to have protoplasts which can be regenerated into plants is of course that protoplasts can be easily transformed or transduced or provided with additional genetic information by any other suitable method. Thus one is now able to provide cassava plants or closely related species with genetic material of interest. The invention thus also provides a method for transforming (defined as providing with in any suitable manner) a protoplast of a cassava or a closely related species by providing said protoplast with additional genetic information through infection by a bacterium comprising said additional genetic information such as Agrobacterium tumefaciens, by electroporation or chemical poration providing a vector comprising said additional genetic information or by particle bombardment whereby the particles are coated with the additional genetic information, whereby a protoplast obtainable from friable embryogenic callus is transformed. The invention also encompasses transformed protoplasts obtainable by such a method.
Below a short introduction is given on the usefulness of transforming plants, such as cassava.
Application of plant gene technology encompasses a multitude of different techniques ranging from isolation of useful genes, their characterization and manipulation, to the reintroduction of modified constructs into the plants (Lonsdale, 1987). Plant gene technology will catalyze progress in plant breeding, as is exemplified by a few examples of transgenic crops like rice (Chen et al., 1987; Shimamoto et al., 1989), maize (Gordon-Kamm et al., 1990; Vain et al., 1993), wheat (Marks et al., 1989), and potato (De Block, 1988; Visser et al., 1989). Rapid progress in gene technology has allowed insight into the complex molecular mechanism of plant pathogen recognition and the natural defence strategies of host plants. This technology can also be used for controlled and efficient identification of desirable genotypes, far beyond the possibilities of classical breeding.
For instance electroporation of protoplasts derived from suspension cultures led to the transformation of maize (Rhodes et al., 1988), rice (Toriyama et al., 1988) and orchardgrass (Horn et al., 1988).
Successful attempts have been made to improve resistance against pathogenic viruses like tobamovirus in tobacco (Powel Abel et al., 1986), potexvirus in potato (Hoekema et al., 1989) and in papaya (Fitch et al., 1992). In the above examples the introduced trait was based on expressing of single genes that are coding for the coat protein. In cassava, African cassava mosaic virus (ACMV) and cassava common mosaic virus (CCMV) may be controlled by the coat protein-mediated resistance technique (Fauquet et al., 1992). The genes encoding key enzymes of cyanogenesis have been cloned (Hughes et al., 1994) which makes manipulation of cassava cyanogenesis by genetic transformation using the antisense approach feasible. Another embodiment of the invention is the manipulation of starch in the cassava tubers.
Thus the present invention provides a transformed protoplast whereby the additional genetic information comprises an antisense construct, particularly one whereby the antisense construct is capable of inhibiting the amylose synthesis pathway.
A protoplast cannot grow in the field, nor can it be harvested. Though protoplasts are necessary for transformation, it must be possible to regenerate said protoplasts into embryos and/or plants. This is a very important embodiment of the invention, because cassava has been shown to be difficult to regenerate from protoplasts. The detailed description explains how this may be achieved. For further information reference is made to the thesis written by E. Sofiari titled Regeneration and Transformation of Cassava (Manihot Esculenta Crantz.), a copy of which is enclosed with the present application, which is as yet unpublished and which is incorporated herein by reference. Thus the invention provides a method for regenerating plants from protoplasts whereby a protoplast according to the invention is induced to produce an embryo, which embryo is consequently induced to produce a plant.
The plants obtainable by said method are also part of the invention, in particular plants whereby the tubers contain essentially no amylose.